Secretion efficiency in Saccharomyces cerevisiae of bovine pancreatic trypsin inhibitor mutants lacking disulfide bonds is correlated with thermodynamic stability.
Bovine pancreatic trypsin inhibitor (BPTI) has been widely used as a model protein to investigate protein structure and folding pathways. To study the role of its three disulfide bonds in folding, proofreading, and secretion of BPTI in an intact eucaryotic cell, BPTI was expressed and secreted from a synthetic gene in the yeast Saccharomyces cerevisiae. Site-directed mutagenesis was used to create all possible single and pairwise cysteine to alanine BPTI mutants, and the effect of these mutations on secretion efficiency was determined. The 5-55 disulfide bond is found to be essential for secretion-loss of either Cys5, Cys55, or both prevents secretion. Removal of the 14-38 disulfide bond results in a small reduction of secretion, but individual Cys14 or Cys38 replacements reduce secretion efficiency by 30%. Cys30 and Cys30-51 mutants are secreted at half the level of wild-type BPTI, while secretion of the Cys51 mutant is reduced by 90%. BPTI containing only a single disulfide bond (5-55) is not secreted. No relationship is observed between secretion efficiency and in vitro folding or unfolding rates, but mutant BPTI secretion is directly correlated with the in vitro unfolding temperature Tm and the free energy of stabilization provided by each of the three disulfides. These results indicate that structural fluctuations rather than the time-averaged structure observed by NMR or X-ray crystallography may determine recognition of a protein as misfolded and subsequent retention and degradation.
Kowalski
J M
JM
Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
Parekh
R N
RN
Wittrup
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D007611:Aprotinin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002417:Cattle; D004220:Disulfides; D005786:Gene Expression Regulation; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
D004220:Disulfides; D011994:Recombinant Proteins; D007611:Aprotinin; D019704:Protein Disulfide-Isomerases